This application claims priority of a German patent application 100 24 404.1 filed May 19, 2000 which is incorporated by reference herein.
The present invention concerns a method and an apparatus for scanning a specimen with a light beam of a light source, preferably in confocal scanning microscopy, the light beam being deflected with a beam deflection device and the scanning operation being controlled by a control device.
In confocal scanning microscopy, a specimen is scanned with a focused light beam; this is generally achieved by tilting two mirrors arranged in the beam path of the confocal scanning microscope. The focus of the light beam is thereby moved in the focal plane, the deflection directions of the light beam most often being arranged perpendicular to one another so that, for example, one mirror deflects the beam in the X direction and another mirror deflects the beam in the Y direction. The motion or tilting of the mirrors is usually brought about with the aid of galvanometer actuating elements.
The intensity of the light coming from the specimen is measured at definable time intervals during scanning; to generate an image, the light intensity value of a specimen point must be unequivocally allocated to the associated scan position of the light beam. For this purpose, the status data for the adjusting elements of the mirrors, i.e. the galvanometer actuating elements, is usually also continuously measured.
In many applications, especially in physiology, the specimen must be observed with a confocal scanning microscope during or shortly after an external influence. These operations are in most cases highly time-critical, so that the instant of the influence must be synchronized with the scanning operation or an image taken by the confocal scanning microscope. The purpose of the physiological applications is, for example, to apply voltage using the xe2x80x9cpatch-clampxe2x80x9d technique to the specimen being detected, or to inject chemicals, so as to detect and analyze the specimen""s reaction to the influence, during or immediately after the influence, using confocal images.
A direct synchronization of the scanning operation of commercially available confocal scanning microscopes with an external influence on the specimen being examined has not hitherto been provided. As an alternative, an internal electrical line synchronization signal of the control device of the confocal scanning microscope is used to generate a synchronization signal usable for the purpose. This merely makes a signal available to a user at the beginning of each scan line. It is not possible with this synchronization method to ascertain directly when the light beam actually reaches the location in the relevant specimen region being externally influenced. Instead, the user must have a further logic unit count the number of lines until the light beam arrives at the corresponding line of the relevant specimen region. He or she must furthermore have a calculation made, again by an external logic unit, of the time required for the light beam to travel from the beginning of the line to the relevant specimen region. This procedure entails considerable difficulty, however, for example because the scanning speed of the light beam is generally variable and this must be taken into account by the external logic unit. A delay value must therefore be calculated as a function of the scanning speed of the light beam and the corresponding line number or local coordinate. This value must be added to the signal of the first line of an image so that the influence on the specimen can be exerted at the time when the light beam will actually reach the relevant specimen region.
It is therefore the object of the present invention to describe a method for scanning a specimen with a light beam of a light source, preferably in confocal scanning microscope, with which a specimen can be scanned, with the greatest possibly accuracy in time, in order to trigger a measurement operation during or shortly after an external influence.
The above object is achieved by a method for scanning a specimen with a light beam of a light source comprising the steps of:
deflecting the light beam with a beam deflection device;
controlling scanning operation by a control device;
providing by the control device, as a function of at least one definable scan position, at least one signal; and
influencing thereby the specimen and/or for triggering a measurement operation.
It is a further object of the present invention to provide an apparatus for scanning a specimen with a light beam of a light source, preferably in confocal scanning microscope, with which a specimen can be scanned, with the greatest possibly accuracy in time, in order to trigger a measurement operation during or shortly after an external influence.
The above object is accomplished an apparatus for scanning a specimen with a light beam of a light source, comprising:
a beam deflection device for deflecting the light beam;
a control device for controlling the scanning operation and providing at least one definable scan position and the control device makes available a signal for influencing the specimen and for triggering a measurement operation; and
a logic unit for compensation for differences in the transit time of the scanning light beam.
What has been recognized according to the present invention is firstly that synchronization of the external influence on the specimen and of a measurement operation can be simplified if a signal for influencing the specimen or for triggering the measurement operation is made available by the control device at exactly the correct time. The external logic unit thus becomes superfluous, thus advantageously making possible a direct coupling, for example, between the unit for influencing the specimen and the confocal scanning microscope. In particular, the user no longer needs to program or configure the logic unit, for which purpose the internal details of the change over time in the scanning operation of the light beam must be known, to say nothing of the considerable electronics-related and hardware-related programming knowledge needed for the purpose.
According to the present invention, the control device makes the signal available as a function of at least one definable scan position. This definable scan position could be, for example, exactly the image point that is to be scanned during or after an external influence on the specimen, so that an observation of the reaction of the specimen to the influence is possible by way of an accurately timed image. In very general terms, the definable scan position is a relevant specimen region. Since the control device controls the specimen scanning operation, the control device can be configured in such a way that it also ascertains the instant at which the light beam arrives at the definable scan position.
The local coordinates of the definable scan position could be stipulated on the basis of detected specimen data. For example, a specimen for microinjection could be prepared with a corresponding microinjection apparatus, and this preparation could be performed in a conventional transmitted-light microscopy mode. A confocal scanning microscope image of the specimen would then be detected, the designated focal plane being the specimen plane in which the tip of the microinjection needle is located. On the basis of the detected confocal scanning microscope image, the tip of the microinjection needle could be exactly stipulated as the definable scan position.
Stipulation of the local coordinates of the definable scan position could be accomplished automatically or interactively. For example, the image of the tip of the microinjection needle could be automatically recognized on the basis of its pattern, using a pattern recognition algorithm. Alternatively, the user could interactively stipulate the local coordinates of the definable scan position. It is similarly conceivable, after the automatic determination of a definable scan position, for an additional further definable scan position to be accomplished interactively. For example, in addition to the automatically determined definable scan position (the tip of the microinjection needle), a further definable scan position could be accomplished interactively by the user. For example, a specimen point that has a functional relationship to the microinjection operation could be selected as a further definable scan position. In this example, a signal is thus made available by the control device on the one hand when the scanning light beam reaches the position of the microinjection needle, and on the other hand when the light beam reaches the specimen point.
Interactive stipulation of the local coordinates of the definable scan position is accomplished with the aid of a pointing device, preferably with a computer mouse connected to the control computer of the confocal scanning microscope.
The stipulated local coordinates of the definable scan position are transferred preferably in digitized fashion to the control unit. The definable scan position at which, for example, provision is made for influencing the specimen is then therefore known to the control unit.
Provision is also made for the present position of the light beam to be sensed. For this purpose, for example, the beam deflection device could make available position data about the present position of the light beam. Those position data could be converted into electronically processable data, or digitized. The position data thus rendered electronically processable could be converted into electronically processable data, or digitized. In a subsequent method step, the position data would be conveyed to the control device.
The elapsed time until the signal is made available, or the elapsed time until the light beam reaches the definable scan position, could similarly be converted into electronically processable data, or digitized. Digitization should involve at least 16 bits, since the time spans to be digitized, ranging from seconds to nanoseconds, must be digitally represented with correspondingly high resolution.
In a concrete method step, provision is made for a logic unit to perform the calculation of the elapsed time until the signal is made available, or the elapsed time until the light beam reaches the definable scan position, based on the scanning speed of the light beam and the local coordinates of the definable scan position. This logic unit is preferably associated with the control device, but it could also be incorporated into the control device. Particular scanning modes of the light beam, for example multiple line averaging, are also taken into account by the logic unit. Provision is additionally made for the present position of the light beam to be taken into account in the calculation of the elapsed time.
Provision is furthermore made for a comparison between the position data for the light beam during scanning and the position datum for the definable scan position. Provision is additionally made for a comparison between the actual position of the beam deflection device (i.e. of the light beam) and the reference position defined by the control device, which represents an important prerequisite in particular for highly accurate synchronization. This method step, too, could be performed by a logic unit that is also incorporated into the control device.
In particularly advantageous fashion, the control unit makes the signal for influencing the specimen and/or for triggering a measurement operation available before and/or after the light beam reaches the definable scan position. Particularly if the control device makes the signal available before the light beam reaches the definable scan position, it is possible thereby to compensate for a corresponding delay time, which corresponds, for example, to the reaction/activation time of the apparatus for influencing the specimen. Especially in xe2x80x9ccaged compoundxe2x80x9d applications in which the influence on the specimen is brought about by the light beam itself, it can be greatly advantageous if the signal is not made available until the light beam has reached the definable scan position. In this case, for example, a measurement of the change in the electrical potential of a cell membrane could be triggered, i.e. a measurement operation could be performed at a well-defined time.
In particularly advantageous fashion, a time span is defined between the arrival of the light beam at the definable scan position and the provision of the signal. This definition can take into account a possible activation time of the apparatus for influencing the specimen, specifically when the apparatus for influencing the specimen requires a certain time in order ultimately to influence the specimen. In this case the time span would need to be selected in such a way that the control device makes the signal available before the light beam reaches the definable scan position.
For a number of applications, provision is made for the signal of the control device to be made available as the light beam reaches the definable scan position. This would make possible exact synchronization of the light beam with the influencing of the specimen and/or the triggering of a measurement operation. The range within which a time discrepancy between the two eventsxe2x80x94the arrival of the light beam at the definable scan position and the provision of the signalxe2x80x94can be tolerated depends on the individual case, and defines a time window which in turn defines simultaneity for the corresponding application.
The control device preferably makes an electrical signal available. An optical or acoustic signal would also be conceivable. An optical signal could, for example, be important in terms of zero-potential transfer of the signal, especially in patch-clamp applications, since in this context, inter alia, very small voltages and/or currents of the specimen are measured, and electrical decoupling of the confocal scanning microscope from the apparatus for influencing or measuring the specimen represents an important parameter for this purpose.
The apparatus according to the present invention for scanning a specimen with a light beam of a light source can, in this context, be implemented in a confocal scanning microscope, the light beam being deflectable with a beam deflection device. The beam deflection device usually comprises one or two mirrors that are pivoted or rotated by galvanometer actuating elements. In the context of a confocal scanning microscope, the scanning operation is generally controlled by a control device.
According to the present invention, the control device makes available, as a function of at least one definable scan position, a signal for influencing the specimen and/or for triggering a measurement operation. The confocal scanning microscope according to the present invention accordingly has an electronic and/or optical interface at which the signal for influencing the specimen or for triggering a measurement operation can be picked off for a corresponding influencing or measuring device.
In a preferred embodiment, in the interest of high timing accuracy a concrete logic unit, which is capable of compensating for differences in the transit time of the scanning beam, is provided. This logic unit is preferably associated with the control device, since the control device usually also makes available the position data about the instantaneous position of the light beam from the beam deflection unit.
In particularly advantageous fashion, data processing in the logic unit and in the control device is accomplished with field-programmable gate arrays (FPGAs). An FPGA is a programmable digital electronic device with which a considerable processing speed can be achieved with quasi-hardware data processing. At present, for example, up to approximately 250 MIPS (million instructions per second) can be executed, thus making possible information processing even at high light beam scanning speeds. A substantial advantage of using FPGAs, however, is their absolute real-time capability, which makes possible reproducible processing with a guaranteed response in the nanosecond range.
In particularly advantageous fashion, a high light beam scanning rate is achievable because of this high timing accuracy of the control device or logic unit equipped with FPGAs. As a result, it is advantageously possible for even very short-duration eventsxe2x80x94or specimen reactions to an external influencexe2x80x94to be not only detected in accurately timed fashion but also documented and logged with sufficient time resolution. Data processing is accordingly accomplished at least almost in real time.